The translation initiation factor eIF5B is an ortholog of the prokaryotic translation factor IF2. Whereas the primary function of IF2 is thought to be binding the initiator methionyl-tRNA to ribosomes, eIF5B promotes ribosomal subunit joining. The eIF5B is a ribosome-dependent GTPase. The three-dimensional structure of eIF5B from the archaeon M thermoautotrophicum was solved by x-ray crystallography. The protein resembles a molecular chalice with the GTP binding domain forming the cup, a long rigid alpha-helix as the stem, and a small beta-barrel domain as the base of the chalice. Comparison of active, GTP-bound and inactive, GDP-bound eIF5B revealed that minor changes in the structure of the GTP-binding domain were amplified through a lever-type mechanism involving the long alpha-helix and resulted in significant movement of the C-terminal beta-barrel domain. Mutational analysis of the GTP-binding domain of yeast eIF5B revealed a critical role for GTP-binding and hydrolysis by eIF5B for translation initiation. Using an eIF5B mutant that utilizes XTP in place of GTP, we have demonstrated that at least two nucleotide (GTP) hydrolysis events are required for eukaryotic translation initiation. Consistent with a role in subunit joining, yeast strains lacking eIF5B show increased levels of leaking scanning. The eukaryotic translation factor eIF1A is an ortholog of the prokaryotic factor IF1, that is known to bind to the ribosomal A-site. We have found that eIF5B and eIF1A physically and functionally interact to promote protein synthesis and yeast cell growth. This evolutionarily conserved interaction between eIF5B/IF2 and eIF1A/IF1 may facilitate initiator methionyl-tRNA binding to the ribosomal P site. Finally, GTP hydrolysis and the accompanying domain rearrangements in eIF5B may promote factor release, and Met-tRNA and ribosomal adjustments that enable subunit joining. The binding of initiator methionyl-tRNA to ribosomes is catalyzed in eukaryotic organisms by the heterotrimeric factor eIF2. The mammalian kinases PKR, HRI, and PERK and the yeast kinase GCN2 specifically phosphorylate serine-51 on the alpha subunit of eIF2 to regulate translation during stress conditions. We demonstrated that the vaccinia virus K3L and swine pox virus C8L proteins are pseudosubstrate inhibitors of PKR, and can suppress PKR function in both yeast and mammalian cells. This inhibition of PKR by K3L and C8L was dependent on residues conserved among eIF2alpha, K3L and C8L. PKR mutants resistant to K3L inhibition are currently under investigation. Whereas an isolated PKR kinase domain was inactive in both yeast and mammalian cells, fusion of heterologous dimerization domains to the PKR kinase domain restored activity. Of particular note, a GyrB-PKR kinase domain fusion protein was found to phosphorylate eIF2alpha and inhibit reporter gene expression in mammalian cells dependent on the drug coumermycin, which promotes GyrB dimerization. Mutational analysis of yeast eIF2alpha revealed a stringent requirement for residues 1-200 for phosphorylation of Ser-51. Amino acid substitutions at residues 49 and 50 as well as in a conserved sequence motif around 30 residues C terminal of the Ser-51 phosphorylation site impaired translational regulation. Biochemical studies revealed that a subset of the mutations in eIF2alpha blocked phosphorylation by the GCN2 and PKR kinases both in vivo and in vitro. These results demonstrate that kinase recognition of eIF2alpha utilizes residues both nearby and, surprisingly, remote from the phosphorylation site.